The present invention relates to compositions and methods for screening compounds that modulate meiotic progression and spermatogenesis. In particular, it relates to compositions comprising nucleotides from the mouse 5xe2x80x2 regulatory region, and transcriptionally active fragments thereof, that control the expression of a testis-enriched protein, SP-10. Specifically provided are expression vectors, host cells, and transgenic animals, wherein the SP-10 promoter controls the expression of a heterologous gene, e.g., green fluorescent protein. The invention relates to methods for using said vectors, cells, and animals for screening candidate molecules for agonists and antagonists of sperm development and fertilization. Methods for using molecules and compounds identified by the screening assays for therapeutic treatments are provided.
Meiotic arrest in spermatogenesis is a well-documented cause of infertility in men. The early stages of spermatogenesis are key steps in the terminal differentiation of male germ cells. Therefore, proteins expressed in early spermatogenesis are likely to be important targets for agonists and antagonists of fertilization. To date, no transcriptional regulatory elements or trans-acting factors involved in the early round spermatid gene expression have been identified. One problem has been the absence of an in vitro model system that recapitulates spermatogenesis. Such a model for terminal differentiation of male germ cells could be used to identify and characterize transcriptional regulators of spermiogenesis. Such regulators could then be targeted to screen for novel compounds and drugs that can be used as treatments for infertility and as contraceptives.
Spermatogenesis is a complex and continuous process of germ cell differentiation which consists of three phases: a proliferative phase, involving the spermatogonial stem cells; a meiotic phase, in which the spermatocytes undergo reduction divisions; and spermiogenesis during which the haploid spermatids undergo extensive biochemical and morphological changes to become spermatozoa. This progression of undifferentiated spermatogonia into spermatozoa is dependent upon precise, developmental stage-specific and germ cell type-specific gene expression.
The 5xe2x80x2 flanking sequences for a limited number of testis-specific genes have been cloned, and the minimal promoters required for testis-specific gene expression have been defined in transgenic mice. These include phosphoglycerate kinase (Pgk-2), proenkephalin, acrosin, histone Hit, private dehydrogenase (Pdha-2), and lactate dehydrogenase (Ldhc-4) genes (Robinson et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:8437-8441; Galcheva-Gargova, 1993, Mol. Endocrinol., 7:979-991; Nayernia et al., 1994, J. Biol. Chem. 51:32181-32186; Bartell et al., 1996, J. Biol. Chem. 271:4046-4054; Iannello et al., 1997, Mol. Cell. Biol. 17:612-619; Li et al., 1998, J. Biol. Chem. 273:31191-31194) which are expressed in the spermatocytes during meiotic phase, and protamine 1, protamine 2, and testis angiotensin converting enzyme (t-ACE) genes (Peschon et al., 1987, Proc. Natl. Acad. Sci. USA. 84:5316-5319; Stewart et al., 1988, Mol. Cell. Biol. 8:1748-1755; Langford et al., 1991, J. Biol. Chem. 266:15559-15562) which are expressed in the late spermatids.
Promoter analysis of genes uniquely expressed in early round spermatids, however, has not been previously reported. SP-10 is an intra-acrosomal protein first identified in human sperm (Herr, 1990, Biol. Reprod. 42:377-382) and subsequently shown to be present in the acrosome of monkey, baboon (Freemerman et al., 1993, Mol. Repr. Dev. 34:140-148), fox (Beaton et al., 1995, Mol. Repr. Dev. 40:242-252.), mouse (Reddi et al., 1995, Biol. Reprod. 53:873-881), and bull (Olson et al., 1997, Biol. Reprod. 57:325-30 334) sperm. Following the acrosome reaction, some amount of SP-10 protein is retained on the inner acrosomal membrane (Foster et al., 1994, Biol. Reprod. 51:1222-1231).
The finding that anti-SP-10 antibodies block sperm-egg interactions in vitro (Coonrod et al., 1996, J. Reprod. Fert. 107:287-297) suggested a role for SP-10 during fertilization. In the human seminiferous epithelium, SP-10 mRNA was first detected in early Golgi phase step 1 spermatids during formation of the acrosomal granule (Kurth et al., 1993, Anat. Rec. 236:619-625). An extensive tissue specificity analysis indicated that SP-10 transcription is testis-specific (Freemerman et al., 1994, Biol. Reprod. 50:615-621).
The invention disclosed herein, based on the identification of SP-10 gene sequences, provides a useful model for the characterization and modulation of testis-specific gene transcription during acrosome biogenesis in round spermatids.
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The invention disclosed herein provides a model for testis-specific gene transcription during round spermatid development. The invention is based in part on the functional characterization described herein of the novel SP-10 promoter, the first testis-specific gene promoter found to be active in early round spermatids.
The present invention provides compositions and methods for screening compounds that modulate meiotic progression and spermatogenesis. In particular, it provides compositions comprising nucleotides from the mouse SP-10 5xe2x80x2 regulatory region, and transcriptionally active fragments thereof, as well as nucleic acids that hybridize under highly stringent conditions to such nucleotides, that control the expression of a testis-enriched protein, SP-10. Specifically provided are expression vectors comprising the SP-10 5xe2x80x2 regulatory region, and transcriptionally active fragments thereof, operably associated to a heterologous reporter gene, e.g., green fluorescent protein (GFP), and host cells and transgenic animals containing such vectors. The invention also provides to methods for using such vectors, cells, and animals for screening candidate molecules for agonists and antagonists of sperm development, which will in turn act as modulators of fertility. Methods for using molecules and compounds identified by the screening assays for therapeutic treatments are provided.
For example, and not by way of limitation, a composition comprising a reporter gene is operatively linked to an SP-10 gene regulatory sequence, herein called the SP-10 promoter. The SP-10 driven reporter gene is expressed as a transgene in mice. The transgenic mice, and cells derived from the testes of such transgenic mice, can be used to screen compounds for candidates useful for modulating development of the haploid round spermatid. Without being bound by any particular theory, such molecules are likely to interfere with the function of trans-acting factors, such as transcription factors as well as cis-acting elements, such as promoters and enhancers required for early spermatogenesis. As such, they are potentially powerful candidates for fertility treatment and for use as contraceptives.
In one embodiment, the invention provides methods for high throughput screening of compounds that modulate sperm differentiation. In this aspect of the invention, testicular cells are removed from the transgenic mice and cultured in vitro. The expression of the reporter gene is used to monitor testes-specific gene activity. In a specific embodiment, green fluorescent protein (GFP) is the reporter gene. Compounds identified by this method can be tested further for their effect on fertilization in normal mice.
In another embodiment, the transgenic mouse model of the invention can be used for in vivo screening to test the mechanism of action of candidate fertility drugs and contraceptive agents and their effect on spermatogenesis. Specifically, the effects of contraceptive agents on terminal differentiation of spermatids into sperm, i.e., spermiogenesis, can be assayed.
In another embodiment the invention provides therapies for targeting defects in meiotic progression leading to azospermia and infertility. Compounds that interfere with acrosomal biogenesis, which marks the beginning of the terminal phase of male germ cell differentiation, can provide therapies for targeting defects in spermatogenesis leading to azospermia and infertility.
In another embodiment, spermatogenic cells derived from transgenic mice are used as a source for transplantation experiments. In various embodiments, transplanted spermatogenic cell SP-10-driven reporter genes are used as markers in assays to test the effect of conditions and factors on various stages of spermiogenesis.
In another embodiment, a gene therapy method for contraception is provided. SP-10 promoter sequences are used to drive spermatid-specific expression of drugs or toxins and introduced in the testis. The method comprises introducing an SP-10 regulatory sequence operatively associated with a drug or toxin gene into testicular cells. In a specific embodiment, the invention provides a gene therapy method for treatment of cancer or other proliferative disorder. The SP-10 promoter is used to direct the expression of one or more proteins specifically in testicular tumor cells of a patient.
The invention further provides methods for screening for novel transcription factors that modulate the SP-10 promoter. Such novel transcription factors identified by this method can be used as targets for male contraceptive discovery.